Construction ofClostridium butyricum hybrid plasmids and transfer toBacillus subtilis

Summary Small plasmids were isolated from type strains ofClostridium butyricum. Strain NCIB 7423 carries one plasmid (pCBU1) of 6.4 kb, whereas strain NCTC 7423 carries two unrelated plasmids of 6.3 kb (pCBU2) and 8.4 kb (pCBU3). Cleavage sites for 18 restriction endonucleases have been mapped on these plasmids and detailed physical maps are presented. For the purpose of developing vector plasmids for gene cloning in saccharolytic clostridia these crypticC. butyricum plasmids were joined to a selectable marker that will likely be expressed in clostridia. This was achieved by cloning the clostridial plasmids into theE. coli vector pBR322 carrying the chloramphenicol acetyltransferase (CAT) gene from theStaphylococcus aureus plasmid pC194. The recombinant plasmids were tested for their ability to confer chloramphenicol resistance toBacillus subtilis. Hybrid plasmids (pHL105, pHL1051) derived from pCBU2 were identified, which are capable of replication and expression of theS. aureus drug resistance marker in bothE. coli andB. subtilis. No structural instability was detected upon retransformation of pHL105 fromB. subtilis intoE. coli. The recombinant plasmids might thus be useful as shuttle vectors for the gene transfer betweenE. coli and a wide range of bacilli and clostridia.     

Analysis of the intracellular DNA-protein associations inEscherichia coli andBacillus subtilis

Intracellular DNA-protein complexes free of RNA have been isolated fromEscherichia coli B andBacillus subtilis 168. The complexes were characterized by the protein/DNA ratio (approximately 0.4) and by physico-chemical parameters. Using electrophoretic methods, it was shown that the protein component of the studied complexes from both microorganisms contained acid and basic proteins. The composition of the protein component of complex isolated fromBacillus subtilis was studied with respect to the growth rate of the culture. It was found that the sample from a slowly growing culture contained always higher amounts of basic proteins with a lower electrophoretic mobility than that from a culture growing more rapidly. A possible role of these proteins is discussed.     

Construction and characterization of hybrid plasmids containing the Escherichia coli nrd region.

Recombinant plasmids containing all or part of the genetic region of Escherichia coli coding for the two subunits of ribonucleoside diphosphate reductase (proteins B1 and B2) were constructed with the aid of the multicopy plasmid pBR322. Two of these plasmids (pPS1 and pPS2) appeared to carry both a regulator and the complete structural information for the enzyme and, after transformation of E. coli, directed a 10- to 20-fold overproduction of both proteins B1 and B2. The other plasmids (pPS101 and pPS201) carried structural information for only protein B2. Cells carrying pPS1 and pPS2 showed a 5- to 500-fold increased resistance against the drug hydroxyurea. This establishes that in E. coli the inhibition of deoxyribonucleic acid synthesis by hydroxyurea is fully explained by its action on ribonucleotide reductase.     

Conjugal transfer of R plasmids to and from Enterobacteriaceae isolated from sewage

A. FERNANDEZ-ASTORGA, A. FERNANDEZ DE ARANGUIZ, M. POCINO, A. UMARAN AND R. CISTERNA. 1992. The potential of the transfer of natural plasmids between sewage strains has been studied. In vitro transfer was conducted at 37°C in tryptone soya broth and sterile raw sewage as mating media. In situ transfer was carried out in sterile raw sewage within membrane diffusion chambers at 10.6°C. When the recipient was a laboratory strain of Escherichia coli K-12, the in situ frequency values were significantly lower ( P < 0.001) than those obtained in vitro for the same mating pair. When the laboratory recipient was replaced with recipients from the same sewage source, frequency values decreased progressively from the optimum conditions to the most adverse. However, in situ frequency values were higher than those for the same donors mated with a laboratory recipient.     

Effect of salinity on the adaptive capacity of recombinant strains ofEscherichia coli andBacillus subtilis

The effect of different concentrations of salts on natural and recombinant strains ofBacillus subtilis andEscherichia coli was studied. The recombinant strain ofB. subtilis was found to be more osmotolerant than the wild-type strain of this bacterium, whereas the opposite situation was observed for the recombinant and wild-type strains ofE. coli. Some salts exerted a bacteriostatic effect onE. coli andB. subtilis. The adaptive capacity of recombinant strains depended on the number of plasmid copies in the cells. The introduction of recombinant bacteria into model ecosystems resulted in the generation of their variants with increased osmotolerance.     

Construction of a colony bank of E. coli containing hybrid plasmids representative of the Bacillus subtilis 168 genome. Expression of functions harbored by the recombinant plasmids in B. subtilis.

Summary A collection of about 2500 clones containing hybrid plasmids representative of nearly the entire genome of B. subtilis 168 was established in E. coli SK1592 by using the poly(dA)·poly(dT) joining method with randomly sheared DNA fragments and plasmid pHV33, a bifunctional vector which can replicate in both E. coli and B. subtilis. Detection of cloned recombinant DNA molecules was based on the insertional inactivation of the Tc gene occurring at the unique BamHI cleavage site present in the vector plasmid.Thirty individual clones of the collection were shown to hybridize specifically with a B. subtilis rRNA probe. CCC-recombinant plasmids extracted from E. coli were pooled in lots of 100 and used to transform auxotrophic mutants of B. subtilis 168. Complementation of these auxotrophic mutations was observed for several markers such as thr, leuA, hisA, glyB and purB. In several cases, markers carried by the recombinant plasmids were lost from the plasmid and integrated into the chromosomal DNA. Loss of genetic markers from the hybrid plasmids did not occur when a rec ^- recipient strain of B. subtilis was used.Abbreviations Ap^R resistance to ampicillin - Tc^R resistance to tetracycline - Cm^R resistance to chloramphenicol - CCC covalently closed circular duplex - Mdal magadalton     

Construction and properties of hybrid plasmids carrying the E. coli gal operon.

A family of hybrid plasmids carrying the entire gal operon of E. coli and designated pgal was constructed in vitro. In the case of pgal 1 (mol. wt. 16.4 Md), a fragment cut by Bam HI endonuclease from lambda gal phage DNA (lambda D-J-gal-att-int) was joined to pMB9 and cloned in the gal-strain of E. coli, which was grown on selective media with galactose as a sole source of carbon. Plasmid pgal2 was derived from pgal 1 by elimination of the 1.1 Md fragment located between the two EcoRI sites and carrying the lambda att-int region and part of pMB9. To obtain pgal3, the 10.7 Md fragment of lambda DNA located between the two SmaI sites (lambda D-J and part of pMB9) in pgal2 was cut out and the resulting flush-end fragments were sealed by the T4DNA ligase. The mol. wt. of pgal3 containing one SmaI site amounted to 4.6 Md, while several pgal3 variants that had lost their SmaI site were still smaller. Plasmid pgal1 inhibited the growth of the gal- host cells, which effect could be overcome by the accompanying helper pMB9. The presence of pgal2 and pgal3 supported the growth and multiplication of gal- cells on selective media even without the helper plasmid. The total amount of pgal plasmid DNA per cell was constant and equalled 60--70 Md (4 copies of pgal1 or 15--16 copies of pgal3, ColE1 or pMB9). This might explain why the co-presence of pMB9 helper does alleviate the "harmful" effects of the plasmid pgal1 (which carries att-int genes), by reducing the copy number of the latter from four to one.     

Conjugal transfer of R plasmids to and from Enterobacteriaceae isolated from sewage.

The potential of the transfer of natural plasmids between sewage strains has been studied. In vitro transfer was conducted at 37 degrees C in tryptone soya broth and sterile raw sewage as mating media. In situ transfer was carried out in sterile raw sewage within membrane diffusion chambers at 10.6 degrees C. When the recipient was a laboratory strain of Escherichia coli K-12, the in situ frequency values were significantly lower (P less than 0.001) than those obtained in vitro for the same mating pair. When the laboratory recipient was replaced with recipients from the same sewage source, frequency values decreased progressively from the optimum conditions to the most adverse. However, in situ frequency values were higher than those for the same donors mated with a laboratory recipient.     

Molecular comparisons of plasmids isolated from colicinogenic strains of Escherichia coli

The plasmid content of 14 colicinogenic strains of Escherichia coli has been examined. Four strains contained miniplasmids (1.2-2.0 kb). Small plasmids (4-7 kb) were detected in all the strains specifying group A colicins (colicins A, E1, E2, E3 and K; group I plasmids). Larger plasmids (55-130 kb) were detected in seven out of nine strains specifying group B colicins (colicins B, H, Ia, Ib, M, V and S1; group II plasmids). DNA-DNA hybridization with group II plasmids showed a wide variation in the degree of DNA sequence homology among its members. In contrast little (if any) DNA sequence homology was detected with the group I plasmids when the same group II plasmid DNAs were used as hybridization probes. The results of DNA-DNA hybridization studies with the two small group II plasmids (pcolG-CA46 and pcolD-CA23) suggested that these plasmids are equivalent to deleted forms of larger group II plasmids. Our hybridization data thus support the division of colicin plasmids into the two groups (I and II) that have been previously defined from genetic and physiological studies.     

Construction of hybrid plasmids containing the lysA gene of Escherichia coli: studies of expression in Escherichia coli and Saccharomyces cerevisiae

Summary The lysA gene of Escherichia coli has been cloned from a transducing phage on various plasmids, present in different copy numbers in bacterial cells. Synthesis of the product of this gene, diaminopimelate (DAP)-decarboxylase, and its regulation have been studied. Expression does not follow a simple gene dosage effect, maximal expression already being obtained with a six-copy plasmid. This result suggests that either a positive or an autogenous regulatory mechanism is involved. We also used one of the hybrid plasmids to look for expression of the bacterial lysA gene in Saccharomyces cerevisiae. The results indicate that the product of the E. coli gene is not actively translated in yeast.     

Drug resistance and R plasmids in Escherichia coli strains isolated from broilers

In 1978, 1,021 Escherichia coli strains were isolated from 105 field broilers (F) and 1,058 strains from 106 broilers in a zootechnical experiment station (Z), and their drug-resistance patterns and the presence of conjugative R plasmids were compared. The resistance markers examined were tetracycline (TC), chloramphenicol (CM), streptomycin (SM), sulfonamides (SA), kanamycin (KM), and ampicillin (APC). The populations of individuals that excreted resistant strains were 100% in F and 58% in Z. Frequencies of isolation of drug-resistant strains among the total isolates were 93% in F and 36% in Z, indicating that the resistant strains are a rather high proportion of the intestinal flora in F but are slightly less prevalent in Z. The resistance pattern to (TC.SM.SA.KM) was seen at the highest frequency in both groups. Conjugative R plasmids were demonstrated more frequently in field broilers (F). The results reflect the wide use of antibiotics in the livestock industry, resulting in the appearance of drug-resistant strains mostly due to the presence of R plasmids.     

IncH plasmids in Escherichia coli strains isolated from broiler chicken carcasses

Plasmids conferring tellurite resistance were transferred at low temperature (27 degrees C) from Escherichia coli strains isolated from chicken carcasses at the time of slaughter and after storage. They belonged to group IncH, as evidenced by their large molecular weight and incompatibility with plasmid pIP233. E. coli strains contaminating chickens meat can thus represent a source of IncH plasmids in the food chain of humans.     

Transfer of transformation and conjugation plasmids from Escherichia coli to Bacillus subtilis and Azospirillum brasilense]

The conjugative transfer of RP4 plasmid from Escherichia coli to Azospirillum brasilense was detected after introduction and subsequent incubation of these microorganisms in soil. The plasmid transfer via transformation from Escherichia coli to Bacillus subtilis was observed in case both bacteria were growing together in sand containing sucrose solution. The possible reason for low frequency interspecies plasmid transformation under conditions close to natural habitats is poor survival of "domesticated" rather than wild type Bacillus subtilis strains and lack of competence state in this case.     

The influence of pH and external H+ concentration on caesium toxicity and accumulation inEscherichia coli andBacillus subtilis

Summary Toxicity screening ofEscherichia coli NCIB 9484 andBacillus subtilis 007, NCIB 168 and NCIB 1650 has shown Cs⁺ to be the most toxic Group 1 metal cation. However, toxicity and accumulation of Cs⁺ by the bacteria was affected by two main external factors; pH and the presence of other monovalent cations, particularly K⁺. Over the pH range 6–9 bothE. coli andB. subtilis showed increasing sensitivity towards caesium as the pH was raised. The presence of K⁺ and Na⁺ in the laboratory media used lowered caesium toxicity and lowered acumulation of the metal. In order to assess accurately Cs⁺ toxicity towards the bacterial strains it was therefore necessary to define the K⁺:Cs⁺ ratio in the external medium. The minimum inhibitory K⁺:Cs⁺ concentration ratio for theBacillus strains tested was in the range 12–13 whileE. coli had a minimum inhibitory K⁺:Cs⁺ concentration ratio of 16.     

Construction of hybrid plasmids containing the Escherichia coli uxaB gene: analysis of its regulation and direction of transcription

The uxaB gene of Escherichia coli, encoding for altronate oxidoreductase involved in the hexuronate degradative pathway, was isolated on a ColE1-uxaB hybrid plasmid from the Clarke and Carbon bank. The restriction map of this plasmid was established. The uxaB gene was mapped on a 1.5-megadalton HindIII-KpnI DNA fragment. Use of an in vitro gene fusion between uxaB and lacZ genes led to the determination that uxaB is transcribed from the KpnI towards the HindIII restriction sites. Gene amplification in cells containing various uxaB hybrid plasmids allowed us to show a gradation in the level of repression of exu operator sites by the exuR regulatory gene product.